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Scientia Horticulturae 129 (2011) 176–182

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Scientia Horticulturae

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actors affecting in vitro adventitious shoot formation on internode explants ofitrus aurantium L. cv. Brazilian

atália T. Marques ∗, Gustavo B. Nolasco, José P. LeitãoIOFIG, Universidade do Algarve, FCT-Edif.8, Campus de Gambelas, 8005-139 Faro, Portugal

r t i c l e i n f o

rticle history:eceived 28 September 2010eceived in revised form 5 March 2011ccepted 13 March 2011

eywords:itrusour orange

a b s t r a c t

The in vitro formation of newly formed adventitious buds and shoots from internodal branch segmentswas studied on 12-month-old plants of Citrus aurantium L. cv. Brazilian. The effects of 6-Benzyladenine(BA) and �-Naphthalene acetic acid (NAA) treatments were evaluated on adventitious bud and shootregeneration. High rates of bud initiation and shoot development were obtained both with BA supple-mented medium, in the range from 1 mg L−1 to 3 mg L−1, and with 0.1 mg L−1 NAA supplemented medium.NAA concentrations above 1 mg L−1 significantly reduced bud initiation and shoot elongation. The resultsobtained using different in vitro culture vessels such as Petri dishes, tubes and glass culture jars were

dventitious budsdventitious shootshoot regenerationrganogenesis

compared. The highest adventitious bud induction was observed in Petri dishes for internodes culturedin 2 mg L−1 BA supplemented medium, with 95% responsive explants forming 9.0 ± 2.4 adventitious buds.The adventitious buds observed in Petri dishes reached a maximum height of 1 mm, with no further devel-opment, while some of the adventitious shoots cultured in tubes and glass culture jars grew over 1 cmin height. A shoot regeneration gradient of the internodes collected along the branch axis was noticed,with basal ones exhibiting higher regeneration frequency.

. Introduction

Citrus aurantium L. cv. Brazilian is a rootstock successfully usedy the Mediterranean’s citrus industry for its excellent qualitiesuch as vigour, disease resistance to soil-borne pathogens, anddaptation to less favourable soil conditions, e.g. calcareous soils.he susceptibility of this rootstock to Citrus tristeza virus (CTV), aajor worldwide threat to Citrus cultures, forced its progressive

ubstitution mainly by the less vigorous CTV-resistant Carrizo cit-ange or Troyer citrange. The increased dissemination of CTV severetrains in the Mediterranean basin (Cerni et al., 2009; Papayiannist al., 2007) and the presence of the most efficient vector Toxopteraitricidus, found in Portugal and North of Spain (Ilharco et al., 2005),ndanger the Mediterranean citriculture and increase the possi-ility of a devastating epidemics (Roistacher and Moreno, 1991).

hus, a deep research with the purpose of merging the excellentgronomical properties of sour orange with resistance to CTV is oftmost importance for the Mediterranean citriculture.

Abbreviations: BA, 6-Benzyladenine; BNS, Basic nutrient solution; IAA, indole--acetic acid; IBA, indole-3-butyric acid; MS, Murashige and Skoog (1962); NAA,-Naphthalene acetic acid; PGR, plant growth regulator; SDS, sodium dodecyl sul-hate.∗ Corresponding author. Tel.: +351 289800100; fax: +351 289818419.

E-mail addresses: nmarques@ualg.pt (N.T. Marques), gnolasco@ualg.ptG.B. Nolasco), jleitao@ualg.pt (J.P. Leitão).

304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.scienta.2011.03.018

© 2011 Elsevier B.V. All rights reserved.

Citrus scion and rootstock cultivars possess features like along juvenile period and a high heterozygosity that prevent Cit-rus improvement through conventional breeding (Cameron andFrost, 1968; Frost and Soost, 1968; Spiegel-Roy and Goldschmidt,1996). As alternative, biotechnological techniques such as plant tis-sue culture and genetic transformation approaches are looked asa valuable strategy for Citrus improvement. Genetic transforma-tion of Citrus is a promising tool that enables the introduction ofdesirable traits without altering the genetic background.

C. aurantium L. has already been the target of genetic trans-formation via Agrobacterium tumefaciens (Ghorbel et al., 2000;Gutiérrez et al., 1997). A low (3.6 ± 1%) percentage of transformedplants were obtained when the CTV CP gene was used (Ghorbelet al., 2000). However, the difficulties encountered with C. auran-tium L. were not only related to the gene transfer process, but alsorelated to a low efficiency of adventitious shoot formation (Ghorbelet al., 1998; Tavano et al., 2009).

The success of a Citrus regeneration protocol relies on a varietyof factors, including the medium used, hormone addendum, incu-bation conditions such as day length, light regime or photon fluxdensity (Bordón et al., 2000; Duran-Vila et al., 1989; Khan et al.,2009; Molina et al., 2007; Moreira-Dias et al., 2000, 2001) and the

explants surface cultured onto the culture medium (García-Luiset al., 2006), without disregarding the enormous influence of geno-type (Barlass and Skene, 1982; Bordón et al., 2000; Moore, 1986;Rodríguez et al., 2008). Notwithstanding the achievements derived

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N.T. Marques et al. / Scientia Horticulturae 129 (2011) 176–182 177

Table 1In vitro shoot regeneration in Citrus aurantium internodes, after 10 weeks of culture, when cultured in: (i) D1–D13 media, (ii) Petri dishes, glass tubes and culture jars.Mean number of buds per cultured explants (MNB/CE) and per responsive explants (MNB/RE). Mean number of buds and shoots per cultured explants (MNBS/CE) and perresponsive explants (MNBS/RE). Percentage of responsive explants (PRE). Mean number of shoots over 1 mm high (MNS > 1 mm) formed per inoculated explant.

Medium BA (mg L−1) NAA (mg L−1) Petri dishes Glass tubes and culture jars

MNB/CEa PRE (MNB/RE) MNBS/CE PRE (MNBS/RE) MNS > 1 mm

D1 – – 0.55 ± 0.3a 39 (1.4 ± 0.1) 1.47 ± 0.2a 70 (2.1 ± 0.4) 0.92 ± 0.1D2 1.0 – 6.27 ± 0.7a 90 (7.0 ± 0.4) 6.71 ± 2.8a 93 (7.1 ± 2.3) 3.07 ± 1.7D3 2.0 – 8.67 ± 2.9a 95 (9.0 ± 2.4) 2.43 ± 1.8a 64 (3.6 ± 2.3) 0.14 ± 0.2D4 3.0 – 7.50 ± 2.9a 83 (8.8 ± 2.3) 1.55 ± 0.3a 41 (3.8 ± 0.0) 0.55 ± 1.3D5 1.0 0.1 8.58 ± 1.5a 95 (9.0 ± 0.9) 4.06 ± 0.3a 75 (5.4 ± 0.4) 2.25 ± 0.2D6 2.0 0.1 8.10 ± 2.5a 90 (8.9 ± 1.4) 4.14 ± 1.4a 60 (8.5 ± 0.7) 1.88 ± 1.0D7 3.0 0.1 5.54 ± 3.1a 71 (7.5 ± 3.0) 3.00 ± 0.9a 50 (5.9 ± 1.0) 0.33 ± 0.2D8 1.0 1.0 4.45 ± 1.1a 92 (6.2 ± 2.5) 1.13 ± 0.9a 56 (2.1 ± 0.4) 0.90 ± 0.7D9 2.0 1.0 3.15 ± 0.5a 85 (3.8 ± 0.4) 1.50 ± 0.2a 75 (2.0 ± 0.3) 0.50 ± 0.2D10 3.0 1.0 3.25 ± 2.8a 75 (4.0 ± 2.8) 0.63 ± 0.5a 63 (1.1 ± 0.1) 0.50 ± 0.3D11 1.0 2.0 1.47 ± 1.2a 27 (5.6 ± 0.5) 0.75 ± 0.2a 50 (1.5 ± 0.2) 0.75 ± 0.2D12 2.0 2.0 0.70 ± 0.3a 37 (1.9 ± 0.1) 1.46 ± 0.1a 55 (3.6 ± 1.9) 1.38 ± 0.1D13 3.0 2.0 2.05 ± 1.1a 54 (3.9 ± 2.3) 1.57 ± 0.2a 79 (2.1 ± 0.1) 0.93 ± 0.1

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ata is from two independent experiments. Values are presented as mean ± S.D. Mukey’s test (P = 0.05).a Mean values are from the 1st and 2nd segment internodes, with ca. 20–30 expl

rom the aforementioned investigations, little research has beenone concerning the effects the type of culture vessel used hasn the in vitro studies of Citrus adventitious shoot induction fromnternodes or epicotyls. Shoot regeneration studies have commonlyeen performed using Petri dishes (Bespalhok Filho et al., 2001;oh et al., 1995; Kobayashi et al., 2003; Tavano et al., 2009), glass

ubes, glass culture jars, or similar vessels (Barlass and Skene, 1982;ordón et al., 2000; García-Luis et al., 1999, 2006; Ghorbel et al.,998; Molina et al., 2007; Moreira-Dias et al., 2001; Pérez-Molphe-alch and Ochoa-Alejo, 1997). Nevertheless, some studies still lack

nformation on the type of culture vessel employed (Maggon andingh, 1995; Moore, 1986).

The use of highly responsive plant material is crucial to the shootegeneration process in order to achieve an elevated number ofdventitious shoots. In Citrus species, including sour orange, intern-des from seedlings less than 2 months old are generally used asxplants (Bespalhok Filho et al., 2001; Goh et al., 1995; Maggon andingh, 1995; Molina et al., 2007; Moreira-Dias et al., 2000, 2001;oore, 1986). Mature tissues have also been tested with lower

egeneration potential. Duran-Vila et al. (1989) referred the use oftem explants of three Citrus species [Citrus aurantifolia (Christm.wing), Citrus medica L. and Citrus sinensis (L.) Osb.]. On C. auran-ium L., Ghorbel et al. (1998) reported the use of 1 cm long stemxplants excised from 12-month-old plants to induce shoot regen-ration. Although a stem offers the advantage of producing severalnternode explants, it is also possible that explants collected alonghe stem respond differentially to the culture medium.

This study reports the research on adventitious shoot regenera-ion in internodes of newly developed branches from 12-month-old. aurantium L. cv. Brazilian plants. In order to determine the effi-iency of the in vitro regeneration process, different concentrationsf 6-Benzyladenine (BA) and �-Naphthalene acetic acid (NAA) plantrowth regulators (PGRs) were tested. It was compared the in vitroud and shoot formation from two distinct segments of the intern-de, along with examination of the shoot regeneration ability ofnternodes along the branch axis. The effect of culture containersPetri dishes, glass culture jars and tubes) on shoot regenerationnd shoot expansion was also evaluated.

. Materials and methods

.1. Plant material

Experiments were performed using 12-month-old plants of C.urantium L. cv. Brazilian. Seeds of sour orange cv. Brazilian were

lues in each column followed by the same letter are not statistically significant by

r each tested medium.

supplied by local agricultural services. The latter were immersedfor 20 min in a 5% (v:v) commercial bleach solution for disinfec-tion and rinsed three times with sterile distilled water. Seeds weresown in peat:vermiculite (1:1). Following germination, seedlingswere transferred to bags containing 25% peat, 45% pine rind, 30%sand, 2.5 g L−1 calcium superphosphate and 3 g L−1 osmocote. Theseeds were then kept in a greenhouse under controlled tempera-ture and with 60–70% humidity. After having been cultured for tenmonths, terminal buds were trimmed to promote the developmentof lateral branches. Newly formed branches, approximately 30 cmlong, were stripped of leaves and thorns, and then submitted to two30 min disinfections, initially with a 5% (v:v) commercial bleachsolution, and afterwards with an extra 0.1% of sodium dodecylsulphate (SDS). This was followed by two rinse steps with ster-ile water. A total of 14 internode explants were numerated fromthe base to the apex of the branch axis. One cm long internodebranch segments were cut transversely just below the node andcultured in a vertical position with the apical end protruding fromthe medium to promote direct regeneration. Further studies wereperformed with 1 cm long internode segments cut immediatelybelow the first internode section. Shoot regeneration responses todifferent plant growth regulator (PGR) concentrations and intern-ode position along the branch axis were analysed in the previouslymentioned explants.

2.2. Culture media and incubation conditions

The basal nutrient medium used contained the salts and vita-mins of “Basic nutrient solution”, a medium previously reported byDuran-Vila et al. (1989), although with an increased concentrationof sucrose (50 g L−1). This basal medium referred to as medium Dwas supplemented with different concentrations of BA and NAAPGRs, according to Table 1. PGRs were added to the medium priorto autoclaving. The pH of the medium was adjusted to 5.7 with 1 MKOH. The culture media, autoclaved for 15 min at 121 ◦C, were thenaliquoted into: 150 mm × 25 mm culture tubes, 90 mm × 15 mmdisposable polystyrene Petri dishes and 80 mm × 90 mm glass cul-ture jars, 25 mL, 30 mL and 50 mL, respectively. Petri dishes andall culture vessels were sterile as well as their polypropylene caps.Petri dishes were sealed with Parafilm® (M Barrier Film). The mediawere solidified with 8 g L−1 Difco Bacto-agar. One explant was

inserted per tube and 5–6 explants were placed per Petri dish orglass culture jar. The explants were maintained in the dark for thefirst 20 days at 25 ± 1 ◦C. Following this period, the explants werekept at the same temperature and exposed to cool white fluores-

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ent light (irradiance 120 mmol m−2 s−1) using a 16/8 h light/darkegime. The explants were subcultured at 4 week intervals. Thevolution of the explants and the number of adventitious shootsormed was recorded after 8–10 weeks culture.

.3. Micrografting and minigrafting

.3.1. MicrograftingSeeds of Carrizo citrange were disinfected by a 30 min immer-

ion in a 0.5% NaOH solution followed by 2–3 wash steps with sterileistilled water. One seed was deposited in each 150 mm × 25 mmube containing sterilized Murashige and Skoog’s (MS) mediumithout organic compounds. MS medium preparation is described

n Section 2.2. Germination occurred in the dark at 27 ◦C. Theicrografting of 0.2–0.5 cm in height regenerated shoots and the

ulture of grafted Carrizo citrange seedlings were performed asescribed by Navarro (1981). Six week-grown micrografted adven-itious shoots with a pair of leaves were grafted onto a one-year-oldigorous rootstock Citrus macrophylla Wester. The grafted plantsere covered with a transparent perforated plastic cap, to maintainumidity, and kept in a climate chamber for 15 days at 25 ± 1 ◦C.hey were then transferred to the greenhouse.

.3.2. MinigraftingSeeds of C. macrophylla Wester were disinfected as previ-

usly described for the Carrizo’s citrange seeds and sown inots containing perlite:peat:vermiculite (1:1:1). One-month-oldeedlings were transplanted into pots containing a mixture ofeat:vermiculite (2:1). After 4–5 months of growth, approximatelycm of the apical stem region was removed and a 3–5 mm deep ver-

ical incision was made on the bark’s previously cut surface in ordero insert an adventitious shoot with more than 0.5 cm in height (Deasquale et al., 1999; Hartmann et al., 1990). The basal portion ofhe adventitious shoot was also cut in a V-shape to facilitate inser-ion into the rootstock incision. The rootstock’s bark was slightlyifted to enable the adventitious shoot’s contact with the cambiumegion. Parafilm® was used to hold the graft in place and a per-orated plastic cap was used to maintain humidity. The T graftingrocess was also performed according to Hartmann et al. (1990).lants were kept in a climate chamber for 2 weeks, at 23–25 ◦C,nd then transferred to the greenhouse while still protected with aerforated plastic cap to maintain humidity.

.4. Experimental design and statistical analysis

.4.1. Effects of BA and NAA concentration on adventitious shootormation

Three concentrations of BA (1, 2 and 3 mg L−1) alone or in com-ination with NAA (0.1, 1 and 2 mg L−1) were tested. The internodeegments were cut from the 7th internode, at the center of theranch until the 14th internode, on the apex. Internode explantsere placed in glass culture vessels. Each BA–NAA concentration

ombination was tested in an equal number of branch internodes.

.4.2. Effect of culture vessel type on adventitious bud and shootormation

A study about the influence of different culture vessels such asetri dishes, tubes and glass culture jars on shoot development waserformed simultaneously with the study on the effects of BA andAA concentration on adventitious shoot formation. The cultureedia and incubation conditions were exactly the same, allow-

ng a comparison between the two studies’ results in terms of the

ffect of the culture vessel employed. The newly formed branch’snternode segments were cut from the 7th internode, at the medianegion of the branch until the 14th internode, on the apex. Intern-de explants were placed in distinct culture vessels and numbered

ulturae 129 (2011) 176–182

according to internode position along the branch axis. The firstand second segments of each internode were also cultured in Petridishes to compare regeneration frequencies. Each combination ofBA and NAA was tested in an equal number of segments from thesame internode. Experiments were performed as duplicates using10–15 segments for each BA and NAA combination and each typeof culture container.

2.4.3. Shoot regeneration from distinct internodes along thebranch axis

Each internode of the branch axis was checked for shoot regen-eration. The regeneration ability of the 1 cm long first and secondsegments was compared for each internode. These segments werecut from the 1st internode, at the base of the branch, until the13th internode. The 14th internode had insufficient height to becut into the two necessary segments to evaluate the segment posi-tion. About 20–30 segments of each segment position were tested.Explants were cultured in medium D5 containing 1 mg L−1 BAand 0.1 mg L−1 NAA. The incubation conditions were the same asdescribed before in Section 2.2. Experiments were performed induplicate.

2.4.4. Measurements and statistical analysisThe number of adventitious buds and shoots was recorded, the

sum of which represents the number of buds & shoots formed. Theregeneration response of the internode explants was evaluated bydetermining: (i) the mean number of buds per cultured explant(MNB/CE) and per responsive explant (MNB/RE); (ii) the meannumber of buds and shoots per cultured explant (MNBS/CE) andper responsive explant (MNBS/RE); (iii) the percentage of respon-sive explants (PRE); and (iv) the mean number of shoots over1 mm in height (MNS > 1 mm). Values of MNB/CE and MNB/RE wererecorded only for Petri dishes. The significance of the main param-eters’ effect on bud and shoot regeneration was analysed using theSPSS program (version 18.0.0). The reported data is the mean oftwo experiments. The data was subjected to analysis of variance(ANOVA) and mean was compared using Tukey’s test with a criticalvalue of P < 0.05.

3. Results

3.1. Effect of BA and NAA concentration on adventitious shootformation

Within 18 days of culture in the dark adventitious buds emergeddirectly from the upper cut end of the explants, in all media testedand containers. The highest bud and shoot initiation was observedfor BA supplemented media ranging from 1 mg L−1 to 3 mg L−1

or with an extra 0.1 mg L−1 of NAA (Table 1, Fig. 1a and b). Thepresence of 0.1 mg L−1 of NAA did not influence significantly for-mation of adventitious shoots. However NAA concentrations above1 mg L−1 clearly reduced bud initiation and shoot elongation. Thepronounced development of one or two shoots usually led to a sup-pressed expansion of neighbouring ones (Fig. 1c). The initiation andexpansion of adventitious shoots also occurred (Table 1, Fig. 1b) inthe absence of PGRs (control medium D1).

3.2. Effect of culture vessel type on adventitious bud and shootformation

In vitro regeneration from internodes in the various vesselstested showed distinct results in terms of bud and shoot for-

mation and expansion. The highest values of shoot regenerationwere attained in Petri dishes (Table 1). Regeneration efficiencywas higher with medium D3 having achieved 8.67 ± 2.9 buds perexplants (MNB/CE), 95% of responsive explants (PRE) and 9.0 ± 2.4

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N.T. Marques et al. / Scientia Horticulturae 129 (2011) 176–182 179

Fig. 1. In vitro shoot regeneration in internode explants: (a) numerous shoots develop per explant on medium D5, bar = 0.7 cm; (b) adventitious shoots formation on mediumD restric , bar =c

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1 (control medium), bar = 0.5 cm; (c) the huge development of one or two shootsut surface of internode number 4, cultured on medium D13, with callus formationallus formation, bar = 0.2 cm.

uds per responsive explant (MNB/RE). The difference between therganogenic ability of the 1st and 2nd segments of each intern-de was not statistically significant (P < 0.05) (results not shown),herefore the results presented in Table 1 (for Petri dishes) arehe combined values obtained for both segments. The adventitiousuds observed in Petri dishes reached a maximum height of 1 mm,ith no further development.

Differences in adventitious bud and shoot initiation betweenlass tubes and glass culture jars were not statistically significantP < 0.05) (results not shown), hence the results presented in Table 1re the combined values obtained for these two containers. Opti-al bud and shoot regeneration was achieved on media D2 with

3% of responsive explants (PRE) and 6.71 ± 2.8 buds and shoots perultured explant (MNBS/CE). Higher results of bud and shoot for-ation (8.5 ± 0.7) were observed when media D6 was being used,

owever explants responsiveness was lower (60%) when comparedith medium D2 results. Many adventitious shoots over 0.5 cm

n height developed in tubes and glass culture jars (Fig. 1a and).

cts the expansion of neighbouring ones, bar = 0.8 cm; (d) adventitious buds on the0.1 cm; (e) adventitious buds formed on explants cultured on medium D2, without

Compact white calli (Fig. 1d) were visible on some of theexplants’ cut surface for the majority of the tested combinationsof PGR concentrations. Some explants only developed calli at thecut surface’s margins while others presented an even calli distribu-tion. After having been cultured in the dark for first 20 days bothcalli and adventitious shoots developed simultaneously, which is anindication of the absence of an intermediate callus phase in shootregeneration. Further histological characterization is needed. Evenwith the presence of calli on explant’s cut surface, buds and shootswere arranged as a ring (Fig. 1d). Explants exposed to higher BAconcentrations had higher calli abundance. In medium D1 (con-trol) and D2 the majority of the explants did not exhibit calli ontheir surfaces (Fig. 1e). Explants from the apex’s distal locations,particularly on internodes 1–6, had higher calli abundance.

3.3. Shoot regeneration from internodes along the branch axis

Bud and shoot regeneration from 1st and 2nd internode seg-ments along the new branch showed no significant statistical

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180 N.T. Marques et al. / Scientia Horticulturae 129 (2011) 176–182

Table 2Internode regeneration response along the branch axis. Results are divided into apical and basal explants, evaluated as the mean number of buds and shoots per culturedexplant (MNBS/CE) and the mean number of buds and shoots per responsive explant (MNBS/RE), after 10 weeks culture.

Internode position MNBS/CE MNBS/RE

Controlc Shoots Shoots > 1 mm Shoots Shoots > 1 mm

Apicala 0.12 ± 0.1a 2.46 ± 0.6a 0.57 ± 0.1a 4.18 ± 0.6a 0.98 ± 0.1aBasalb 0.14 ± 0.1a 4.76 ± 0.6ab 1.02 ± 0.1a 7.45 ± 0.8ab 1.59 ± 0.2a

Data is from two independent experiments. Values are presented as mean ± S.D. Mean values in each column followed by the same letter are not statistically significant byTukey’s test (P = 0.05).

a Internodes from the branch’s apical portion are numbered 7–13. Mean values are from all internodes on the apical portion of the branch, with ca. 20–30 explants for eachinternode number.

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c Internodes cultured on hormone free basal medium D.

ifferences (P < 0.05). The results presented in Table 2 and Fig. 2 arehus the combined values obtained for both 1st and 2nd segments.tatistically significant differences (P < 0.05) were observed for thenternode position along the branch axis. Basal internodes (1st toth internodes) formed superior number of adventitious buds andhoots (7.45 ± 0.8) when compared with the apical internodes (8tho 14th internodes) (4.18 ± 0.6) (Table 2, Fig. 2). Adventitious shootxpansion did not change significantly along the branch axis for the3 internodes tested (P < 0.05) (Table 2, Fig. 2).

.4. Minigrafting and micrografting

Preliminary attempts to root shoots in vitro demonstrated thathis genotype is recalcitrant to rooting (2.9% of responsive explants,esults not shown). The Navarro’s micrografting process (1981) waserformed as an alternative to rooting. The latter was successful,

n 60% of cases, on the development of grafted shoots. Shoots over.5 cm in height were successfully (63%) minigrafted directly ontohe strong C. macrophylla Wester. Developed shoots were of normalhenotype.

. Discussion

Combined or separate BA and NAA PGRs play a key role inhe induction of adventitious shoots from Citrus species internodexplants (Bordón et al., 2000; Ghorbel et al., 1998). The optimal

nternode response for C. aurantium L. cultivars was induced within

and 5 mg L−1 of BA, albeit with lower shoot regeneration val-es and independent of the explant’s development stage (Bordónt al., 2000; Ghorbel et al., 1998; Moore, 1986; Tavano et al., 2009).

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ig. 2. Influence of internode position along the branch axis on the number of budsnd shoots (�) or shoots higher than 1 mm (�), formed per inoculated explant. Eachoint represents the average value from two independent experiments, with ca.0–30 explants for each internode number. Vertical bars = S.D.

all internodes on the basal portion of the branch, with ca. 20–30 explants for each

Tavano et al. (2009) reported that C. aurantium L. requires lowerBA concentrations (0.5 mg L−1) than the ones tested in the cur-rent study. Contradictory reports concerning the effects of NAA onC. aurantium L. shoot regeneration can be found in the Literature.Although NAA has been suggested to have no effect on the numberof shoots produced (Moore, 1986), other studies have revealed thatin combination with BA it is fundamental in the process (Bordónet al., 2000). Our study shows that, for all tested containers, theNAA concentration at 1 mg L−1 induces a decrease in the initia-tion of buds and shoots. To our knowledge, other auxins such asacid indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), havealready been tested on other Citrus genotypes (Kitto and Young,1981; Maggon and Singh, 1995), but not yet studied on C. aurantiumL. adventitious shoot regeneration.

Epicotyls and internodal segments have been successfully usedas explants for several Citrus scion and rootstock cultivars. In vitrostudies on shoot regeneration efficiency regarding young epicotylsand stem explants of C. aurantium L. state that mature explantscompetence is lower than young ones’ (Tavano et al., 2009). Theregeneration of adventitious shoots from epicotyl explants of C.aurantium has been previously reported to have: 29% responsiveexplants with 3 ± 2 shoots per cultured explant (Moore, 1986);and 18% of the explants presenting 0.8 ± 0.5 shoots per responsiveexplants (Bordón et al., 2000). A previous study on 12-month-old plant stem internodes reports 60% of responsive explantswith 1.5 ± 0.6 adventitious shoots formed per responsive explant(Ghorbel et al., 1998). Older explants than the epicotyls are thuspromising explants for the in vitro shoot regeneration process. Thisstudy attained a 93% high shoot regeneration (PRE) on 12-month-old explants cultured in medium D2 filled glass jars and tubes, withan average of 6.71 ± 2.8 shoots formed (MNBS/CE). The high valuesof morphogenesis achieved in the present work may be related tothe vitality of the stem segments used, and perhaps in contrastwith the plant material used by Ghorbel et al. (1998), which mayhave been older and more lignified than the vigorous newly formedbranches employed in this study.

The air volume capacity of the culture vessels has been sug-gested to possibly influence the number of shoots formed on eachexplant. A factor suggested to retard adventitious shoot growthis the effect of concrete levels of carbon dioxide, ethylene andother volatile gases that accumulate in the container’s air space(Heo et al., 2006; Leng et al., 2004; Tisserat and Vaughn, 2008).The present study clearly shows that in vitro shoot induction washigher for internodes cultured in Petri dishes, virtually with less airspace than glass tubes and culture jars. Petri dishes were wrappedwith Parafilm®, which allows gas exchange. However, it seems thateventual gas accumulation within the container may explain the

enhanced bud formation observed. On the other hand, gas accumu-lation may be accountable for the restrictive shoot developmentobserved in Petri dishes. It was previously reported that in vitroregenerated shoots from Citrus paradisi’s nodes grew higher in

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ented tubes than in unvented ones (Marutani-Hert et al., 2010).n addition, humidity accumulation inside Petri dishes is expectedue to plants transpiration and low evaporating conditions. Highumidity conditions are associated with high axillary bud multi-lication rate in rose explants (Sallanon and Maziere, 1992). It maylso explain the prolific number of adventitious shoots formed. Theattern of buds and shoot formation in Petri dishes suggests that these of two containers in a two-step process may be advantageousor shoot regeneration: (1) initial bud formation in Petri dishes dur-ng the first month of in vitro culture, followed by (2) subculture onlass jars to promote shoot development. Further experiments toonfirm the latter hypothesis are needed.

A gradient in the organogenic response was reported foreedling explants along the Troyer’s citrange epicotyl axis with aaximal response verified at the base, near the cotyledons (García-

uis et al., 1999; Goh et al., 1995; Moreira-Dias et al., 2001). On thether hand, distal explants from the cotyledon of two distinct Citruspecies (Citrus limonia Osb.; Citrus paradise Macf.) were reported toe more organogenic (Costa et al., 2004). The current work shows ahoot regeneration gradient along the branch axis, but with higherrganogenic response for internodes at the basal portion of theranch. One may speculate that the basal portion of the branchossesses different nutrient or endogenous plant hormone concen-rations when compared with the apical portion, differences that

ay affect adventitious bud induction. The basipetal movementf auxin along stems has been well documented (Kramer et al.,008; Raven et al., 2005), its relation to the results obtained is stillncertain, requiring further analysis.

C. aurantium has already been described to be recalcitranto rooting (Gutiérrez et al., 1997). Alternative minigrafting and

icrografting protocols were explored in this work to rescuehe regenerated shoots. On one hand, the minigrafting procedurevoids the laborious work of in vitro culture associated with theicrografting process, and promotes a rapid growth and develop-ent of the shoots. On the other hand, this protocol imposes that

he adventitious shoots be over 0.5 cm in height to be able to beinigrafted, which is a disadvantage. Minigrafting is applicable to

ther Citrus species and might be able to circumvent the existingifficulties with rooting regenerated shoots.

.1. Conclusions

This study demonstrates that internode segments from 12-onth-old C. aurantium L. cv. Brazilian plants cultured in low

oncentrations of BA supplemented medium have high shootegeneration efficiency. With the appropriate balance of BA andAA PGRs, the large diameter internodes tolerate the developmentf a high number of adventitious shoots. Culture containers usedn the shoot regeneration process may promote distinct responserom the explants. Shoot development was observed only in intern-des cultured in tubes and glass jars. Furthermore, differences inrganogenic potential along the newly formed branch axis wereound. The factors studied allow enhancing the regeneration fre-uency of internode explants from 12-month-old plants. Thesexplants could be used in experiments of genetic transformationiming to improve the efficiency of production of transgenic C.urantium plants.

cknowledgements

The authors acknowledge the Direccão Geral de Agricultura do

lgarve and the technical assistance of Eng◦ Celso Mendes for pro-iding seeds and establishing conditions for the sowing and growthf sour orange cv. Brazilian. This work was financially supportedy the Foundation for Science and Technology, Portugal, under

ulturae 129 (2011) 176–182 181

research contract PTDC/AGR-GPL/099512/2008 and Center for Bio-diversity, Functional and Integrative Genomics (BioFig), Portugal.

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